Over the past 17 years of this grant, we have produced a large number of monoclonal antibodies (mAb) to myeloid-associated antigens expressed on myeloid leukemia cells. These mAb have been used clinically for the diagnosis and therapy of acute myeloid leukemia (AML). One of the mAb, 251, directed to the CD33 antigen specific for myeloid cells, has been incorporated into bispecific antibodies (BsAb) conjugated to either an anti-CD64 or an anti-CD 16 mAb directed to different Fe receptors. These BsAb mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of myeloid leukemia cells by normal monocytes through Fe receptor- mediated processes. We now propose to focus on the further development of these and an additional BsAb, all targeting CD33 and one of three Fe receptors, for clinical application in the therapy of AML. Here, we will develop an additional BsAb using the same anti-CD33 mAb and an anti-CD89 (Fc alpha receptor) mAb. We will directly compare the relative activities of the three BsAb, all targeting CD33, in vitro for mediating ADCC, phagocytosis, and killing of AML progenitor cells (Specific Aim 1). We will also compare the activities of the three BsAb in a murine/human AML xenograft model (Aim 1). These studies will focus on the relative potency of each BsAb with its unique effector cell population (monocytes, neutrophils, or natural killer cells). In Specific Aim 2 we will focus on the biology of the CD33 antigen and the effects of BsAb on its function. Based on preliminary and published data, CD33 has been determined to be a negative regulator of leukemia cell growth. We will study the mechanisms of action of BsAb. We will examine the molecular effects of BsAb binding to CD33, CD 16, CD64, and CD89 antigens and downstream mediators of cellular function in normal and leukemia cells. In Aim 3, we will extend the observations from Aim 2 to studies on cells from patients entered on a Phase I study of anti-CD33xanti-CD64 BsAb. We will test the hypothesis that the levels of expression of CD33 and CD64 predict the effects of BsAb binding in vivo. At completion of the study, rational planning for clinical trials of BsAb in patients with AML with the most promising BsAb targeting the CD33 antigen will be possible.